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Phytosterol profiles of common foods and estimated natural intake of different structures and forms in China Mengmeng Wang, Weisu Huang, Yinzhou Hu, Liangxiao Zhang, Ya-Fang Shao, Meng Wang, Fang Zhang, Ziyan Zhao, Xiaohong Mei, Tao Li, Donghui Wang, Ying Liang, Jing Li, Yining Huang, Liuquan Zhang, Tao Xu, Huaxin Song, Yongheng Zhong, and Baiyi Lu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05009 • Publication Date (Web): 03 Feb 2018 Downloaded from http://pubs.acs.org on February 18, 2018

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Journal of Agricultural and Food Chemistry

Engineering Laboratory of Intelligent Food Technology and Equipment, Key Laboratory for Agro-Products Postharvest Handling of Ministry of Agriculture, Key Laboratory for Agro-Products Nutritional Evaluation of Ministry of Agriculture，Zhejiang Key Laboratory for Agro-Food Processing, Fuli Institute of Food Science Song, Huaxin; Zhejiang University, College of Biosystems Engineering and Food Science Zhejiang University, Hangzhou 310058, China, National Engineering Laboratory of Intelligent Food Technology and Equipment, Key Laboratory for Agro-Products Postharvest Handling of Ministry of Agriculture, Key Laboratory for Agro-Products Nutritional Evaluation of Ministry of Agriculture，Zhejiang Key Laboratory for Agro-Food Processing, Fuli Institute of Food Science Zhong, Yongheng; Zhejiang University, College of Biosystems Engineering and Food Science Zhejiang University, Hangzhou 310058, China, National Engineering Laboratory of Intelligent Food Technology and Equipment, Key Laboratory for Agro-Products Postharvest Handling of Ministry of Agriculture, Key Laboratory for Agro-Products Nutritional Evaluation of Ministry of Agriculture，Zhejiang Key Laboratory for Agro-Food Processing, Fuli Institute of Food Science Lu, Baiyi; Zhejiang University, College of Biosystems Engineering and Food Science Zhejiang University, Hangzhou 310058, China, National Engineering Laboratory of Intelligent Food Technology and Equipment, Key Laboratory for Agro-Products Postharvest Handling of Ministry of Agriculture, Key Laboratory for Agro-Products Nutritional Evaluation of Ministry of Agriculture，Zhejiang Key Laboratory for Agro-Food Processing, Fuli Institute of Food Science

ACS Paragon Plus Environment


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1

Title

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Phytosterol profiles of common foods and estimated natural intake of different

3

structures and forms in China

4

Author names

5

Mengmeng Wanga, Weisu Huangb, Yinzhou Hua, Liangxiao Zhangc, Yafang Shaod,

6

Meng Wange, Fang Zhangf, Ziyan Zhaog, Xiaohong Meih, Tao Lii, Donghui Wangj,

7

Ying Liangk, Jing Lil, Yining Huangb, Liuquan Zhanga, Tao Xua, Huaxin Songa,

8

Yongheng Zhonga, Baiyi Lua*

9

Author Affiliations

10

a

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Hangzhou 310058, China, National Engineering Laboratory of Intelligent Food

12

Technology and Equipment, Key Laboratory for Agro-Products Postharvest Handling

13

of Ministry of Agriculture, Key Laboratory for Agro-Products Nutritional Evaluation

14

of Ministry of Agriculture，Zhejiang Key Laboratory for Agro-Food Processing, Fuli

15

Institute of Food Science

16

b

17

Hangzhou 310018, China

18

c

19

430062, China

20

d

China National Rice Research Institute, Hangzhou 310006, China

21

e

Beijing Research Center for Agricultural Standards and Testing, Beijing 100097,

College of Biosystems Engineering and Food Science Zhejiang University,

Zhejiang Economic & Trade Polytechnic, Department of Applied Technology,

Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan


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Journal of Agricultural and Food Chemistry 2

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China

23

f

Beijing University of Technology, Beijing 100124, China

24

g

Southwest University, Chongqing 400715, China

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h

China agricultural University, Beijing 100083, China

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i

Chinese Academy of Tropical Agricultural Science, Haikou 571101, China

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j

Chinese Academy of Agricultural Sciences Institute of Agro-Products Processing

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Science and Technology, Beijing 100193, China

29

k

30

China

31

l

32

Xingcheng 125100, China

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Corresponding author

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Baiyi Lu.

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College of Biosystems Engineering and Food Science Zhejiang University, No.866

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Yuhangtang Road, Xihu District, Hangzhou, Zhejiang 310058, P. R. China

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E-mail address: [email protected]

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Abstract

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Phytosterols are well-known for their cholesterol-lowering effects, and the structures

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and forms of phytosterols affect their bioactivity. We aimed to illustrate the

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phytosterol profiles in common foods and estimate their natural intake in five

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geographical regions and among different age groups in China. In total, 12

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phytosterols in free and esterified forms of 119 foods from five regions across China

Key Laboratory of Food Quality and Safety of Jiangsu Province, Nanjing 210014

The Research Institute of Pomology, Chinese Academy of Agricultural Sciences,



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were examined using gas chromatography–mass spectrometry. Then, the dietary

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intake of phytosterols was calculated combined with the dietary foods intake data of

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Chinese people. Total Phytosterol content was highest in vegetable oils (150.4–1230.9

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mg/100 g), followed by legumes (129.6−275.6 mg/100 g), nuts (18.9−255.2 mg/100

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g), and cereals (11.9–93.8 mg/100 g). Vegetables and fruits contained lower contents

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of total phytosterols. Phytosterols were mainly esterified in most common food except

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in nuts. The predominant phytosterols were β-sitosterol, campesterol, and stigmasterol,

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all of which belonged to plant sterols and 4-desmethylsterols. Total phytosterol intake

52

varied

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mg/standard-person(sp)/day, with the highest intake in Beijing, followed by

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Hangzhou, Wuhan, Chongqing, and Guangzhou. However, phytosterol proportion was

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similar across regions, with β-sitosterols accounting for 46.5%–50.3% of the natural

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intake.

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4-desmethylsterols in esterified form (61.9%–74.6%). At the age of 2–70 years,

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phytosterol intake ranged from 154.3 mg/day to 348.0 mg/day in the national scale.

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Keywords: phytosterols; profile; dietary intake; β-sitosterol

60

1.

across

different

Phytosterol

regions,

intake

was

ranging

mainly

between

constituted

by

257.7

plant

and

sterols

473.7

and

Introduction

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Phytosterols are compounds whose structure resembles that of cholesterol with

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difference in the side chain, such as β-sitosterol with an ethyl group at C-24,

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campesterol with a methyl group at C-24, or the number of methyl group at C-4 in A

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ring, for example, cylcoartanol with two methyl groups at C-4 1. Phytosterols are


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mainly found in plant and marine organisms and cannot be synthesized in the human

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body, while cholesterols were mainly found in animal. After the 3–4 weeks’

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intervention of daily consumption of 23 g of phytosterols, the serum LDL-cholesterol

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could decline 10%–15%

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disorders. Epidemiological and experimental studies have suggested that dietary

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phytosterols protect from most common cancers in Western societies, such as colon,

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breast, and prostate cancers 5. Over 250 different sterols exist in free or esterified

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forms 1 and can be divided into plant sterols and plant stanols based on their degree of

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saturation, with the latter being the saturated form of the former. Phytosterols can also

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be classified as 4-desmethylsterols and 4,4’-dimethylsterols based on the number of

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methyl groups at C-4. The common 4-desmethysterols includes β-sitosterol,

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campesterol, stigmasterol and brassicasterol, and the common 4,4’-dimethylsterols

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includes 24-methylene cycloartanol, cycloartenol and cycloartanol.

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2-4

, thus conferring protection from potential cardiovascular

The difference in structure and form of phytosterols affects their bioactivity, such 6-8

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as absorption, efficacy of lowering cholesterol levels, and anti-cancer activity

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Generally, plant stanols are less absorbed than plant sterols

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cholesterol levels

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cholesterol-lowering effect while the cholesterol-lowering effect of plant sterols

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would diminish between 1 and 2 month12. 4-desmethylsterols are more effective at

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lowering cholesterol levels than 4,4’-dimethylsterols13. Esterified phytosterols must

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first be hydrolyzed to reduce cholesterol absorption 14. Besides, esterified phytosterols

9-10

.

and equally reduced

11-12

, but O’Neil reported plant stanols could maintain their



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significant reduced the biaoavailability of β-carotene more than that of free

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phytosterols15. Phytosterols content had been reported, for example, in United States

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Department of Agriculture (USDA) National Nutrient Database for Standard

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Reference, individual content of sitosterol, stigmasterol, and campesterol has been

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reported in only 1.5% of foods, without distinguishing between the free and esterified

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forms

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focused on vegetable oils, nuts, and cereals, while little research has been done on

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legumes, tubers, vegetables, and fruits. For example, Phillips et al. reported the free

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and esterified phytosterol content of 31 edible oils used in USA and Finland

95

Piironen et al. reported the free and esterified phytosterol content of several cereals

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used in Finland

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phytosterol content in nuts and seeds 19-20. Furthermore, Han et al. have quantified the

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phytosterol content in common plant-derived foods in China

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evaluated five major phytosterols, without distinguishing between the free or

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esterified form. The amount of free and esterified phytosterol in common foods in

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China is still unknown.

16

. When analyzing free and esterified phytosterols, most published reports

17

, and

18

. Researchers have also analyzed the free, esterified, and total

21

, but they have only

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The recommended daily intake of phytosterols was 2 g by the American Heart

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Association22 and 2–3 g by National Cholesterol Education Program23, while the

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natural daily intakes of phytosterols were reported around 100–400 mg/d in western

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counties24-26. According to Han, the Chinese phytosterols daily intake was 322 mg21.

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However, all the estimation didn’t distinguish the free and esterified form of


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phytosterols. China has a large population and the dietary patterns differed a lot

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among regions. The aims of the present study were to determine the individual

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phytosterol content in free and esterified forms of the common foods, and to estimate

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the phytosterol intake with different structure in free and esterified form of five

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regions and total phytosterols intake of different age groups in China.

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2.

Materials and methods

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To better estimate the effect of different dietary patterns on phytosterol intake,

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we chose the following five large cities that represented the north, south, east, west,

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and center of China, respectively: Beijing, Guangzhou, Hangzhou, Chongqing, and

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Wuhan. In each of the five regions, we first roughly divided the region into five parts

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according to the populations, then chose at least five supermarkets in local to get the

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common food samples. All food samples were obtained from local supermarkets.

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2.1 Standards and reagents

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Stigmasterol (95%), β-sitosterol (≥97%), campesterol (98%), brassicasterol

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(≥95%), ergosterol (98%), campestanol (95%), sitostanol (95%), ∆5-avenasterol

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(95%), α-spinasterol (97%), cycloartanol (97%), cycloartenol (95%), 24-methylene

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cycloartanol

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heptafluorobutyramide (MSHFBA), and 1-methyl imidazole (1-MIM) were purchased

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from Sigma-Aldrich. Acetone (HPLC), ethanol (AR), diethyl ether (AR), hexane

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(HPLC), and dichloromethane (AR) were ordered from Merck & Co, Inc.

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2.2 Lipid extraction

(92%),

5α-cholestane

(≥97%),


N-methyl-N-(trimethylsilyl)


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Cereals, legumes, tubers, and nuts were ground into fine powder and stored at

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−18°C in sealed bags. Vegetables and fruits were dried under reduced pressure and

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then frozen and powdered under liquid nitrogen. Except for oils, the moisture content

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of all the samples was determined and lipids were extracted using Soxhlet extraction

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method of Chinese National Standard GB 5009.6-2016. Each sample was separated

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into two part. One part was (approximately 5 g) was used to determine the moisture

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content and placed in dishes at 103 °C ± 2 °C until constant weight was attained

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(approximately 8 h). The other part (approximately 2–5 g) was weighed into an

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extraction vessel and loaded into an Auto-Fat Determinator SZC-C (Shanghai

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Qianjian, Shanghai, China), then lipids were extracted using 30 mL of petroleum ether,

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which has a boiling point of 30 °C–60 °C (approximately 6 h). After the solvent was

139

evaporated under nitrogen, the lipid content was stored at −20 °C in the dark for

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further treatment.

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2.3 Extraction of free phytosterols

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With some modifications, free phytosterols were analyzed based on the method 27

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reported by Esche et al.

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cartridge (Strata NH2, 55 µm, 70 Å, 1 g/6 mL, Phenomenex, Aschaffenburg,

145

Germany) to activate the column. Then, 50 mg of the crude lipid fraction or vegetable

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oils containing 50 µg of 5α-cholestane as an internal standard was dissolved in 5 mL

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of n-hexane and loaded. The fractions of steryl/stanyl fatty acid esters and interfering

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triglycerides were eluted with 2 × 5 mL of n-hexane/diethyl ether (98:2, v/v) and 4 ×

. Briefly, 2 × 5 mL of n-hexane was loaded onto an SPE


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5 mL of n-hexane/ethyl acetate (96:4, v/v). Next, free sterols/stanols were eluted with

150

2 × 5 mL of n-hexane/ethyl acetate (5:95, v/v), and the solvents were then removed by

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evaporation under nitrogen. The free phytosterols would be analyzed by gas

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chromatography–mass spectrometry.

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2.4 Extraction of total phytosterols

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The detection of total phytosterols was carried out according to the method of M 28

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Rudzinska et al.

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as an internal standard to 20 mg of crude lipid fraction or vegetable oils. Lipids were

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then saponificated with 1 M KOH/ethanol for 18 h at room temperature

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(approximately 25 °C). With water and diethyl ether extracting the unsaponifiables,

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residues were kept after the solvent was removed by nitrogen. The total phytosterols

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would be analyzed by gas chromatography–mass spectrometry.

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2.5 Gas chromatography–mass spectrometry (GC–MS) analysis

with some modifications; e.g., 50 µg of 5α-cholestane was added

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The phytosterols (free phytosterols extracted in section 2.3 and total phytosterols

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extracted in section 2.4) were silylated with 100 µL of MTBSTFA/1-MIM (95:5, v/v)

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at 75°C for 20 min, then dissolved in 1 mL of n-hexane, and analyzed using a

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7890A-5973N GC–MS system (Agilent Technologies, USA) equipped with a

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DB-5MS column (30 m × 0.25 mm × 0.25 µm; Agilent Technologies). The oven

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temperature was initially set at 180°C, held for 1 min, then raised to 290°C at the rate

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of 40°C/min, and again held for 10 min. Nitrogen was used as carrier gas at a rate of

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1.6 mL/min. With the positive electron ionization (EI+) in mass spectrometry, the



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electron energy was set at 70 eV and the ion source temperature at 250 °C. The

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retention time of commercial standards was used to identify phytosterols, and each

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peak was evaluated by the detection of the parent molecular ion and the fragmentation

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pattern of its TMS derivative. The trimethyl silylation samples were detected using

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the Full Scan Mode with m/z = 50–600 and analyzed by the quantitative ions

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(Supplementary Table 1) in SIM mode.

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2.6 Estimation of phytosterol intake

177 178 179

The concentration of phytosterols esters was calculated as the difference of the concentration of total phytosterols minus that of free phytosterols. The daily intake of phytosterols were calculated as follows:

Phytosterols daily intake = phytosterols concentration in food ∗ food daily intake

180

The “phytosterols concentration in food” were from the data we determined. The

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“food daily intakes” data were obtained from “Survey on the Status of Nutrition and

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Health of Chinese people.” Based on this, we calculated the total phytosterol intake in

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five regions of China and across different age groups in the national scale. Next, we

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estimated the intake of phytosterols with different structures and forms, including

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plant sterols and stanols, 4-desmethylsterols and 4,4’-dimethylsterols, and free and

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esterified phytosterols.

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2.7 Statistical analysis

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All analyses were carried out in triplicate, and the results were expressed as the

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mean ± standard deviation. The calculations were performed with SPSS for Windows


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version 20.0. To classify the phytosterols profiles in foods, the heatmap was generated

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in the MetaboAnalyst version 3.5. Figure 1, 2 and 4 were drew in Origin 9.0.

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3. Results

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3.1 Phytosterol profiles in common foods

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3.1.1

Total phytosterols in foods

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The total phytosterol content greatly varied among foods (Supplementary Table

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2). Generally, when calculated as dry weight, vegetables oils, legumes, tubers, and

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nuts were rich in phytosterols, followed by cereals. Vegetables and fruits contained

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less phytosterol content. Figure 1A illustrates the average total phytosterol content of

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oils, nuts, cereals, legumes, vegetables, and fruits. The average total phytosterol levels

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in vegetable oil, tubers, cereals, and nuts were 518.4, 144.2, 130.5, and 99.9 mg/100 g,

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respectively. Vegetables and fruits had the lowest phytosterol content (24.8 and 22.0

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mg/100 g, respectively).

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In vegetable oils, the highest phytosterol content was found in rice oil (1230.9

204

mg/100 g) and the lowest in camellia oil (150.4 mg/100 g). The total phytosterol

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contents of rapeseed, corn, and sesame oils were 878.6, 712.1, and 652.9 mg/100 g,

206

respectively. For linseed, sunflower seed, peanut, soybean, and olive oils, the total

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phytosterol content ranged from 280.0 to 352.9 mg/100 g. The most common tubers

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in China were sweet potato, potato, and Chinese yam, with the average total

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phytosterol content in dry matter as 140.3, 113.8, and 178.5 mg/100 g, respectively.

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Other good sources of phytosterols were legumes (129.6–275.6 mg/100 g) and bean



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products (40.5–69.2 mg/100 g). The total phytosterol content of cereals ranged from

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11.9 to 93.8 mg/100 g. Common cereals can be classified into rice-, wheat-, and other

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cereal-based products as per the Chinese dietary pattern. In general, rice products

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contained the highest phytosterol content among cereals, the average being 54.7

215

mg/100 g. Other cereals had a phytosterol content of 34.3 mg/100 g, whereas

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wheat-based products had the lowest content (23.2 mg/100 g). In nuts, the total

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phytosterol content ranged from 15.2 mg/100 g in chestnuts to 255.2 mg/100 g in

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pistachios. In vegetables and fruits, bamboo shoots contained the highest phytosterol

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levels (95.3 mg/100 g).

220

3.1.2

Free and esterified phytosterols in foods

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The percentage of free phytosterols is shown in Figure 1B. The composition of

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free and esterified phytosterols widely varied among foods, with the esterified form

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being predominant. However, in nuts, 57.0%–76.4% of phytosterols were in the free

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form. Free phytosterols also predominated in some vegetable oils, such as sunflower

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seed, peanut, and soybean (range, 55.2%–59.8%), but were found in less quantities in

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linseed, rice, corn, rapeseed, sesame, olive, and camellia oils (range, 12.5%–42.4%).

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Most cereals contained more esterified phytosterols than free phytosterols, except

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buckwheat grains and corn starch, in which free phytosterols constituted 71.3% and

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63.4%, respectively. In most tubers, legumes, vegetables, and fruits, phytosterols

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mainly existed in esterified forms. Individual phytosterols were in similar percentage

231

in the free and esterified forms.


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3.1.3

Plant sterols and plant stanols in foods

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Plant sterols were the main phytosterols in almost all foods. β-sitosterol was the

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predominant phytosterol in vegetable oils (contributing 32.3%–67.7%), tubers

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(15.9%–34.6%), legumes (13.2%–34.7%), nuts (61.9%–86.7%), cereals (27.2%–

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62.4%), vegetables (0.7%–78.6%), and fruits (0.5%–100.0%). Campesterol and

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stigmasterol were the next predominant phytosterols, although there were some

238

exceptions. For example, rapeseed oil contained more brassicasterol than stigmasterol,

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whereas α-spinasterol was predominant in spinach, amaranth, cucumber, towel gourd,

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winter melon, pumpkin, zucchini pumpkin, and watermelon seed kernels.

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Figure 2 illustrates the distribution of individual phytosterols in vegetable oils,

242

tubers, legumes, nuts, and cereals. In the heat map, we chose Euclidean distance to

243

measure similarity and Ward’s linkage to cluster by minimizing the sum of squares of

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any two clusters. Heat map is an intuitive visualization method to analyze the

245

distribution of experimental data. In Figure 2, Each small square represents each food

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sample, and its color indicates the amount of individual phytosterols. The higher the

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concentation is, the darker the color is (the red is up, the green for down). Each row

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shows the concentration of each phytosterols in different samples, and each column

249

shows the concentration of all the phytosterols in each sample. The upper tree

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represents cluster analysis results for different samples and the left tree represents

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cluster analysis results for different phytosterols from different samples. As shown in

252

the heat map, the foods could be classified into three groups based on the phytosterol



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253

profile. The first group mostly comprised vegetable oils; the second comprised

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legumes, tubers, linseed oil, sunflower seed oil, pistachios, pine nuts, pumpkin seed

255

kernels, and watermelon seed kernels; and the third group comprised cereals and most

256

nuts.

257

3.1.4

4-desmethylsterols and 4,4’-dimethylsterols in foods

258

In the study, the calculations of 4-desmethysterols includes β-sitosterol,

259

campesterol, stigmasterol, ∆5-avenasterol, brassicasterol, α-spinasterol, sitostanol,

260

campestanol, ergosterol, and the calculations of 4,4’-dimethylsterols includes

261

24-methylene cycloartanol, cycloartenol and cycloartanol. In most food groups,

262

4-desmethylsterols were predominant. In vegetable oils, 4,4’-dimethylsterol content

263

ranged from 12.0 to 260.0 mg/100g, with rice bran oil containing the highest levels,

264

whereas in nuts, it ranged from 0.4 to 8.2 mg/100 g, contributing to 2.5%–17.1% of

265

the total phytosterol content. In cereals, the 4,4’-dimethylsterol content was around

266

30.0 mg/100 g in rice products, which was almost equal to that of β-sitosterol. In

267

wheat products and other grains, however, this content was

Phytosterol profiles of common foods and estimated natural intake of

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